Ferrihaemoprotein Hydroxides 1 19 



only about 60-75 % hydroxide formation. It is already evident from Fig. 10, 

 however, that the hydroxide has a pronounced peak at about 540 mn with 

 a second peak at about 575 ra^i, and no peaks either in the region 600 to 

 640 m/i or at about 500 m,a. Spectroscopically, as well as magnetically, it 

 can safely be classified as a low-spin complex. The spectroscopic type is 

 fully substantiated by the corresponding spectrum for Japanese root peroxi- 

 dase (Morita and Kameda, 1958), which has absorption bands at 548 and 

 578 ma, with £niM = 12-3 and lOT respectively, together with relatively 

 lower absorption in the region 620 to 650 m//, compared to horseradish 

 peroxidase in Fig. 10. But its magnetic moment has not yet been measured. 



The data for two other haemoglobins may be considered at this point. 

 The first, Chironomus haemoglobin (Scheler and Fischbach, 1958), presents 

 some anomalous features. The visible spectrum of the hydroxide is most like 

 that of ferrimyoglobin, and the shape of the curve in the region of 600 m,a 

 suggests that it should come between ferrimyoglobin and ferrihaeraoglobin 

 in the sequence in equation (7), but on a quantitative scale nearer the former. 

 However its magnetic moment is 4-45 B.M., a little less than that of ferri- 

 haemoglobin hydroxide (Scheler, Schoffa and Jung, 1957). No explanation 

 can be offered for this discrepancy, although it is to be noted that the magnetic 

 moment of the acidic ferrihaemoglobin is appreciably lower than the values 

 determined for ferrimyoglobin and erythrocyte ferrihaemoglobin, namely 

 5-68 and 5-80 B.M. respectively (Theorell and Ehrenberg, 1951; Coryell, 

 Stitt and Pauling, 1937). 



The second, root nodule haemoglobin (leghaemoglobin) is particularly 

 interesting. The visible spectrum of the hydroxide, reproduced in Fig. 16 

 (Sternberg and Virtanen, 1952), is almost the same as that of Japanese root 

 peroxidase, which indicates that it is a low-spin complex. Furthermore, 

 preliminary spectroscopic observations by George, Hanania and Thorogood 

 (1959) in the near infra-red have shown it to have significantly lower absorp- 

 tion in the region 700 to 900 m,a than the myoglobin and haemoglobin 

 derivatives, which is in keeping with the trend in extinction coefficients from 

 high- to low-spin complexes (see Fig. 7). The magnetic moment however 

 still remains to be determined. Hence, provided it is appropriate to regard 

 leghaemoglobin as a true haemoglobin*, the haemoglobins themselves, 

 without recourse to peroxidase, furnish a series of hydroxides covering almost 

 the whole range of spectroscopic characteristics. 



There is thus a substantial body of evidence to suggest that the hydroxides, 

 especially of ferrimyoglobin and ferrihaemoglobin, are mixtures of high- and 



* This classification is based on the ability of ferroleghaemoglobin to form an oxygen 

 complex, and it is further substantiated by the reaction of ferrileghaemoglobin with 

 hydrogen peroxide. An intermediate compound is formed with absorption bands at 550 

 and 575 m//, resembling the ferrimyoglobin and ferrihaemoglobin derivatives, in contrast 

 to ferriperoxidase and ferricatalase, which give two such compounds neither having bands 

 at these wavelengths. 



